Many aspects of human biology, including cognitive abilities, advanced language, and susceptibility to common diseases, are influenced by genome sequence changes that occurred very recently in human evolution. Genomic sequence from the Neandertal, which most evidence suggests diverged from the modern human lineage -500,000 years ago, would help identify these events. The goal of this study is to obtain Neandertal genome sequences by targeted methods in order to identify fixed sequence changes unique to modern humans. The aims of our proposal are based on extensive preliminary data, including the creation of genomic DNA libraries from Neandertal and cave bear remains, the generation of Neandertal genomic sequences, and the targeted recovery of specific sequences from cave bear genomic libraries. We will first analyze several Neandertal samples available to us to identify those that show the best Neandertal DNA preservation with minimal amounts of modern human contamination. Using these Neandertal samples, we will construct Neandertal metagenomic libraries that will serve as a renewable source of Neandertal genomic DNA for these studies and which we will also make available to the research community at large. We will use these libraries in both direct genomic selection and PCR-based searches for specific Neandertal genomic clones, focusing on sites of human-chimpanzee nucleotide substitution in genes and other functional sequences. The data generated by these methods will be used in an extensive effort to compare human, chimpanzee and Neandertal sequences and identify substitutions that occurred after humans diverged from Neandertals. We will authenticate Neandertal sequences by recovering the same sequences from multiple Neandertal libraries. We will exclude human-Neandertal substitutions polymorphic in modern humans by comparison to known SNPs and by resequencing substituted loci in a diverse panel of human samples. These studies will identify a set of human-chimpanzee sequence differences that are unique to modern humans, yielding fundamental insights into human origins, the rise of human-specific phenotypic traits, and the biology of Neandertals. Lay Summary: Humans are a relatively recent species, arising in the last -160,000 years. However, many aspects of our biology, including our language, our ability to reason, and our susceptibility to many diseases, are unique to us. We hope that by comparing our genome sequence to that of the Neandertal, or closest, but now extinct relative, we will identify the changes that occurred in our genome that led to the rise of unique human traits.